Development and Validation of a Quantitative PCR Assay Using Multiplexed Hydrolysis Probes for Detection and Quantification of Theileria orientalis Isolates and Differentiation of Clinically Relevant Subtypes

Abstract

T heileria orientalis is a vector-borne hemoprotozoan that infects cattle and buffalo and is generally spread by ticks of the Haemaphysalis genus (1-3). Historically, this organism has been referred to as Theileria sergenti, Theileria buffeli, or the T. orientalis/T. sergenti/T. buffeli complex; however, the name T. sergenti is now considered invalid (4), and T. orientalis is commonly used to refer to all (5). From a clinical perspective, T. orientalis can cause anemia, lethargy, jaundice, fever, abortion, and mortality in cattle (6). Clinical infection can also result in decreased milk production in dairy cattle (7). T. orientalis is a conditional pathogen, and while pathogenic forms are largely limited to Eastern Asia and Australasia (5, 6, 8-10), it is frequently detected in asymptomatic animals; these benign forms are globally spread (5,(11)(12)(13)(14). Analysis of the most common genotyping locus (p32), encoding the variable major piroplasm surface protein (MPSP), currently identifies 11 distinct T. orientalis genotypes (13,15,16). Of these genotypes, type 2 (Ikeda) and to a lesser extent type 1 (Chitose) are typically found in association with clinical disease (6,9,10,(17)(18)(19)(20)(21)(22). The presence of pathogenic and benign forms of T. orientalis greatly complicates its clinical diagnosis, with standard blood film analysis unable to identify the pathogenic genotypes.Multiple conventional PCR (cPCR) assays have been published for the identification of T. orientalis in blood samples (9,21,23,24). The most commonly cited assays detect and genotype T. orientalis by amplifying unique regions of the MPSP gene (21). These assays use two universal primers to detect T. orientalis infection and specific forward primers to identify the Ikeda, Chitose, and Buffeli genotypes. While this method is highly sensitive and has been validated in prior studies (19,21), it is not multiplexed and is highly susceptible to PCR inhibitors (6), which are often found in nucleotide extractions obtained from blood (25,26). To overcome inhibition, undiluted and diluted nucleotide extracts can be examined in parallel to prevent false-negative results (6,19,27). However, if multiple reactions per sample are required to determine the presence of infection, the procedure becomes both expensive and time-consuming. Furthermore, cPCR does not give an accurate representation of parasite load and therefore cannot provide an indication of the severity of T. orientalis infection. A recently developed multiplexed tandem PCR (28) is able to discriminate between four genotypes of T. orientalis, but it is only semiquantitative and therefore cannot be used to define a clinical threshold.Hydrolysis probe quantitative PCR (qPCR) assays employ sequence-specific, fluorescently labeled probes attached to duplex-

Temporal dynamics and subpopulation analysis of Theileria orientalis genotypes in cattle

“…All T. orientalis positive samples (n = 137) were derived either from herds with clinical theileriosis cases (n = 89) or from herds with subclinical infections at the time of sampling (n = 48) and were characterised in a previous study as Chitose positive infections (Bogema et al, 2015). Negative samples were sourced from an area where T. orientalis was not enzootic and were confirmed negative by conventional PCR (Bogema et al, 2015).…”

“…Negative samples were sourced from an area where T. orientalis was not enzootic and were confirmed negative by conventional PCR (Bogema et al, 2015). T. orientalis-negative samples sourced from cattle known to be infected with Babesia bigemina or Babesia bovis, causative agents of the clinically similar disease, tick fever; or derived from cattle immunized with Anaplasma centrale, were kindly provided by Dr. Phillip Carter at the Tick Fever Centre, Wacol (Bogema et al, 2015). Time-course samples were collected from a mob of 10 naïve Ayrshire cattle that had been introduced to an area on the midcoast of New South Wales, Australia, where T. orientalis was known to be enzootic, and onto a property with a prior history of clinical theileriosis cases.…”

“…Specificity was also tested using 10 samples derived from T. orientalis-negative animals, or T. orientalis-negative animals known to be positive for B. bovis, B. bigemina or A. centrale. The performance of each assay in detecting its target genotype within clinical samples was also tested using 15 wellcharacterised (sequenced and/or tested with alternate assays) samples known to be positive for one or more of the Ikeda, Chitose and Buffeli genotypes (Bogema et al, 2015;Eamens et al, 2013a).…”

“…All T. orientalis positive samples (n = 137) were derived either from herds with clinical theileriosis cases (n = 89) or from herds with subclinical infections at the time of sampling (n = 48) and were characterised in a previous study as Chitose positive infections (Bogema et al, 2015). Negative samples were sourced from an area where T. orientalis was not enzootic and were confirmed negative by conventional PCR (Bogema et al, 2015). T. orientalis-negative samples sourced from cattle known to be infected with Babesia bigemina or Babesia bovis, causative agents of the clinically similar disease, tick fever; or derived from cattle immunized with Anaplasma centrale, were kindly provided by Dr. Phillip Carter at the Tick Fever Centre, Wacol (Bogema et al, 2015).…”

“…Plasmid DNA standards, reagents, equipment, consumables and thermal cycling parameters for qPCRs performed in this study were as described previously (Bogema et al, 2015) with primers and probes for the Ikeda, Chitose and Buffeli subtype assays as listed in Table 1.…”